A synthetic differentiation circuit in Escherichia coli for suppressing mutant takeover.

Differentiation is crucial for multicellularity. However, it is inherently susceptible to mutant cells that fail to differentiate. These mutants outcompete normal cells by excessive self-renewal. It remains unclear what mechanisms can resist such mutant expansion. Here, we demonstrate a solution by engineering a synthetic differentiation circuit in Escherichia coli that selects against these mutants via a biphasic fitness strategy. The circuit provides tunable production of synthetic analogs of stem, progenitor, and differentiated cells. It resists mutations by coupling differentiation to the production of an essential enzyme, thereby disadvantaging non-differentiating mutants. The circuit selected for and maintained a positive differentiation rate in long-term evolution. Surprisingly, this rate remained constant across vast changes in growth conditions. We found that transit-amplifying cells (fast-growing progenitors) underlie this environmental robustness. Our results provide insight into the stability of differentiation and demonstrate a powerful method for engineering evolutionarily stable multicellular consortia.

Chromatin context-dependent regulation and epigenetic manipulation of prime editing.

We set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing, a precise genome engineering tool. Using a highly sensitive method for mapping the genomic locations of randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% to 94% for an identical target site and edit. Position effects on prime editing efficiency are well predicted by chromatin marks, e.g., positively by H3K79me2 and negatively by H3K9me3. Next, we developed a multiplex perturbational framework to assess the interaction of trans-acting factors with the cis-chromatin environment on editing outcomes. Applying this framework to DNA repair factors, we identify HLTF as a context-dependent repressor of prime editing. Finally, several lines of evidence suggest that active transcriptional elongation enhances prime editing. Consistent with this, we show we can robustly decrease or increase the efficiency of prime editing by preceding it with CRISPR-mediated silencing or activation, respectively.

Structurally divergent and recurrently mutated regions of primate genomes.

We sequenced and assembled using multiple long-read sequencing technologies the genomes of chimpanzee, bonobo, gorilla, orangutan, gibbon, macaque, owl monkey, and marmoset. We identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. We estimate that 819.47 Mbp or ∼27% of the genome has been affected by SVs across primate evolution. We identify 1,607 structurally divergent regions wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (e.g., CARD, C4, and OLAH gene families) and additional lineage-specific genes are generated (e.g., CKAP2, VPS36, ACBD7, and NEK5 paralogs), becoming targets of rapid chromosomal diversification and positive selection (e.g., RGPD gene family). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species.

SnapShot: Skeletal muscle atrophy.

Skeletal muscle size is highly plastic and sensitive to a variety of stimuli. Muscle atrophy occurs as the result of changes in multiple signaling pathways that regulate both protein synthesis and degradation. The signaling pathways that are activated or inhibited depend on the specific stimuli that are altered. To view this SnapShot, open of download the PDF.

Recurrent inversion polymorphisms in humans associate with genetic instability and genomic disorders.

Unlike copy number variants (CNVs), inversions remain an underexplored genetic variation class. By integrating multiple genomic technologies, we discover 729 inversions in 41 human genomes. Approximately 85% of inversions <2 kbp form by twin-priming during L1 retrotransposition; 80% of the larger inversions are balanced and affect twice as many nucleotides as CNVs. Balanced inversions show an excess of common variants, and 72% are flanked by segmental duplications (SDs) or retrotransposons. Since flanking repeats promote non-allelic homologous recombination, we developed complementary approaches to identify recurrent inversion formation. We describe 40 recurrent inversions encompassing 0.6% of the genome, showing inversion rates up to 2.7 × 10-4 per locus per generation. Recurrent inversions exhibit a sex-chromosomal bias and co-localize with genomic disorder critical regions. We propose that inversion recurrence results in an elevated number of heterozygous carriers and structural SD diversity, which increases mutability in the population and predisposes specific haplotypes to disease-causing CNVs.

Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine.

We examined antibody and memory B cell responses longitudinally for ∼9-10 months after primary 2-dose SARS-CoV-2 mRNA vaccination and 3 months after a 3rd dose. Antibody decay stabilized between 6 and 9 months, and antibody quality continued to improve for at least 9 months after 2-dose vaccination. Spike- and RBD-specific memory B cells remained durable over time, and 40%-50% of RBD-specific memory B cells simultaneously bound the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells were efficiently reactivated by a 3rd dose of wild-type vaccine and correlated with the corresponding increase in neutralizing antibody titers. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit the added protection afforded by repeat short interval boosting. These data provide insight into the quantity and quality of mRNA-vaccine-induced immunity over time through 3 or more antigen exposures.

The expanding amyloid family: Structure, stability, function, and pathogenesis.

The hidden world of amyloid biology has suddenly snapped into atomic-level focus, revealing over 80 amyloid protein fibrils, both pathogenic and functional. Unlike globular proteins, amyloid proteins flatten and stack into unbranched fibrils. Stranger still, a single protein sequence can adopt wildly different two-dimensional conformations, yielding distinct fibril polymorphs. Thus, an amyloid protein may define distinct diseases depending on its conformation. At the heart of this conformational variability lies structural frustrations. In functional amyloids, evolution tunes frustration levels to achieve either stability or sensitivity according to the fibril's biological function, accounting for the vast versatility of the amyloid fibril scaffold.

Robust and prototypical immune responses toward COVID-19 vaccine in First Nations peoples are impacted by comorbidities.

High-risk groups, including Indigenous people, are at risk of severe COVID-19. Here we found that Australian First Nations peoples elicit effective immune responses to COVID-19 BNT162b2 vaccination, including neutralizing antibodies, receptor-binding domain (RBD) antibodies, SARS-CoV-2 spike-specific B cells, and CD4+ and CD8+ T cells. In First Nations participants, RBD IgG antibody titers were correlated with body mass index and negatively correlated with age. Reduced RBD antibodies, spike-specific B cells and follicular helper T cells were found in vaccinated participants with chronic conditions (diabetes, renal disease) and were strongly associated with altered glycosylation of IgG and increased interleukin-18 levels in the plasma. These immune perturbations were also found in non-Indigenous people with comorbidities, indicating that they were related to comorbidities rather than ethnicity. However, our study is of a great importance to First Nations peoples who have disproportionate rates of chronic comorbidities and provides evidence of robust immune responses after COVID-19 vaccination in Indigenous people.

The Penium margaritaceum Genome: Hallmarks of the Origins of Land Plants.

The evolutionary features and molecular innovations that enabled plants to first colonize land are not well understood. Here, insights are provided through our report of the genome sequence of the unicellular alga Penium margaritaceum, a member of the Zygnematophyceae, the sister lineage to land plants. The genome has a high proportion of repeat sequences that are associated with massive segmental gene duplications, likely facilitating neofunctionalization. Compared with representatives of earlier diverging algal lineages, P. margaritaceum has expanded repertoires of gene families, signaling networks, and adaptive responses that highlight the evolutionary trajectory toward terrestrialization. These encompass a broad range of physiological processes and protective cellular features, such as flavonoid compounds and large families of modifying enzymes involved in cell wall biosynthesis, assembly, and remodeling. Transcriptome profiling further elucidated adaptations, responses, and selective pressures associated with the semi-terrestrial ecosystems of P. margaritaceum, where a simple body plan would be an advantage.

Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.

Tolerance of infections.

A host has two methods to defend against pathogens: It can clear the pathogens or reduce their impact on health in other ways. The first, resistance, is well studied. Study of the second, which ecologists call tolerance, is in its infancy. Tolerance measures the dose response curve of a host's health in reaction to a pathogen and can be studied in a simple quantitative manner. Such studies hold promise because they point to methods of treating infections that put evolutionary pressures on microbes different from antibiotics and vaccines. Studies of tolerance will provide an improved foundation to describe our interactions with all microbes: pathogenic, commensal, and mutualistic. One obvious mechanism affecting tolerance is the intensity of an immune response; an overly exuberant immune response can cause collateral damage through immune effectors and because of the energy allocated away from other physiological functions. There are potentially many other tolerance mechanisms, and here we systematically describe tolerance using a variety of animal systems.

A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns.

Synthetic multicellular systems hold promise as models for understanding natural development of biofilms and higher organisms and as tools for engineering complex multi-component metabolic pathways and materials. However, such efforts require tools to adhere cells into defined morphologies and patterns, and these tools are currently lacking. Here, we report a 100% genetically encoded synthetic platform for modular cell-cell adhesion in Escherichia coli, which provides control over multicellular self-assembly. Adhesive selectivity is provided by a library of outer membrane-displayed nanobodies and antigens with orthogonal intra-library specificities, while affinity is controlled by intrinsic adhesin affinity, competitive inhibition, and inducible expression. We demonstrate the resulting capabilities for quantitative rational design of well-defined morphologies and patterns through homophilic and heterophilic interactions, lattice-like self-assembly, phase separation, differential adhesion, and sequential layering. Compatible with synthetic biology standards, this adhesion toolbox will enable construction of high-level multicellular designs and shed light on the evolutionary transition to multicellularity.

Structural Studies of Amyloid Proteins at the Molecular Level.

Dozens of proteins are known to convert to the aggregated amyloid state. These include fibrils associated with systemic and neurodegenerative diseases and cancer, functional amyloid fibrils in microorganisms and animals, and many denatured proteins. Amyloid fibrils can be much more stable than other protein assemblies. In contrast to globular proteins, a single protein sequence can aggregate into several distinctly different amyloid structures, termed polymorphs, and a given polymorph can reproduce itself by seeding. Amyloid polymorphs may be the molecular basis of prion strains. Whereas the Protein Data Bank contains some 100,000 globular protein and 3,000 membrane protein structures, only a few dozen amyloid protein structures have been determined, and most of these are short segments of full amyloid-forming proteins. Regardless, these amyloid structures illuminate the architecture of the amyloid state, including its stability and its capacity for formation of polymorphs.

Transcriptome dynamics of CD4+ T cells during malaria maps gradual transit from effector to memory.

The dynamics of CD4+ T cell memory development remain to be examined at genome scale. In malaria-endemic regions, antimalarial chemoprevention protects long after its cessation and associates with effects on CD4+ T cells. We applied single-cell RNA sequencing and computational modelling to track memory development during Plasmodium infection and treatment. In the absence of central memory precursors, two trajectories developed as T helper 1 (TH1) and follicular helper T (TFH) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated TH1 and TFH trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between TFH and central memory were revealed, with antimalarials modulating these responses and boosting TH1 recall. Finally, single-cell epigenomics confirmed that heterogeneity among effectors was partially reset in memory. Thus, the effector-to-memory transition in CD4+ T cells is gradual during malaria and is modulated by antiparasitic drugs. Graphical user interfaces are presented for examining gene-expression dynamics and gene-gene correlations ( http://haquelab.mdhs.unimelb.edu.au/cd4_memory/ ).

Dynamic Control of X Chromosome Conformation and Repression by a Histone H4K20 Demethylase.

Chromatin modification and higher-order chromosome structure play key roles in gene regulation, but their functional interplay in controlling gene expression is elusive. We have discovered the machinery and mechanism underlying the dynamic enrichment of histone modification H4K20me1 on hermaphrodite X chromosomes during C. elegans dosage compensation and demonstrated H4K20me1's pivotal role in regulating higher-order chromosome structure and X-chromosome-wide gene expression. The structure and the activity of the dosage compensation complex (DCC) subunit DPY-21 define a Jumonji demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Selective inactivation of demethylase activity eliminates H4K20me1 enrichment in somatic cells, elevates X-linked gene expression, reduces X chromosome compaction, and disrupts X chromosome conformation by diminishing the formation of topologically associating domains (TADs). Unexpectedly, DPY-21 also associates with autosomes of germ cells in a DCC-independent manner to enrich H4K20me1 and trigger chromosome compaction. Our findings demonstrate the direct link between chromatin modification and higher-order chromosome structure in long-range regulation of gene expression.

A Viral Immunoevasin Controls Innate Immunity by Targeting the Prototypical Natural Killer Cell Receptor Family.

Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via paired NK cell receptors (NKRs). Despite identification of numerous NKR-ligand interactions, physiological ligands for the prototypical NK1.1 orphan receptor remain elusive. Here, we identify a viral ligand for the inhibitory and activating NKR-P1 (NK1.1) receptors. This murine cytomegalovirus (MCMV)-encoded protein, m12, restrains NK cell effector function by directly engaging the inhibitory NKR-P1B receptor. However, m12 also interacts with the activating NKR-P1A/C receptors to counterbalance m12 decoy function. Structural analyses reveal that m12 sequesters a large NKR-P1 surface area via a "polar claw" mechanism. Polymorphisms in, and ablation of, the viral m12 protein and host NKR-P1B/C alleles impact NK cell responses in vivo. Thus, we identify the long-sought foreign ligand for this key immunoregulatory NKR family and reveal how it controls the evolutionary balance of immune recognition during host-pathogen interplay.

Functional Selectivity in Cytokine Signaling Revealed Through a Pathogenic EPO Mutation.

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

Comprehensive Analysis of Hypermutation in Human Cancer.

We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management of patients and families. These data will inform tumor classification, genetic testing, and clinical trial design.

The IRE1 endoplasmic reticulum stress sensor activates natural killer cell immunity in part by regulating c-Myc.

Natural killer (NK) cells are critical mediators of host immunity to pathogens. Here, we demonstrate that the endoplasmic reticulum stress sensor inositol-requiring enzyme 1 (IRE1α) and its substrate transcription factor X-box-binding protein 1 (XBP1) drive NK cell responses against viral infection and tumors in vivo. IRE1α-XBP1 were essential for expansion of activated mouse and human NK cells and are situated downstream of the mammalian target of rapamycin signaling pathway. Transcriptome and chromatin immunoprecipitation analysis revealed c-Myc as a new and direct downstream target of XBP1 for regulation of NK cell proliferation. Genetic ablation or pharmaceutical blockade of IRE1α downregulated c-Myc, and NK cells with c-Myc haploinsufficency phenocopied IRE1α-XBP1 deficiency. c-Myc overexpression largely rescued the proliferation defect in IRE1α-/- NK cells. Like c-Myc, IRE1α-XBP1 also promotes oxidative phosphorylation in NK cells. Overall, our study identifies a IRE1α-XBP1-cMyc axis in NK cell immunity, providing insight into host protection against infection and cancer.

Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system.

In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of RUNX transcription factor paralogs with apparent functional redundancy. Here we asked what cell-type-specific biologies might be supported by the selective expression of RUNX paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional nonequivalence between RUNX paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain and evolutionary reconstruction suggested convergence of RUNT domain residues toward submaximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.